DNA Sequence Encoding a Retinoic Acid Regulated Protein

ABSTRACT

The present invention concerns a novel retinoic acid regulated gene whose expression product displays useful morphogenic/mitogenic properties. The present invention further concerns an isolated nucleic acid of SEQ ID NO:1 encoding a retinoic acid regulated expression product having an amino acid sequence of SEQ ID NO:2.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/339,734, filed Dec. 19, 2008, which is a continuation application ofU.S. application Ser. No. 11/894,091, filed Aug. 20, 2007, nowabandoned, which is a divisional application of U.S. application Ser.No. 10/726,160, filed Dec. 2, 2003, now U.S. Pat. No. 7,276,595, issuedOct. 2, 2007, which is a continuation-in-part application of U.S.application Ser. No. 10/409,511, filed Apr. 8, 2003, now abandoned,which is a divisional application of U.S. application Ser. No.09/354,359, filed Jul. 14, 1999, now abandoned, which are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention is concerned with a novel retinoic acid (RA)regulated gene whose expression product displays usefulmorphogenic/mitogenic properties. In particular, the present inventionis concerned with uses of the expression product. For example, theexpression product may be used generally as a tumor biomarker or as anindicator for Hepatocellular carcinoma (HCC). The marker or indicatorcan also serve as a screening and supporting tool for the diagnosis oftumor such as HCC, and is also useful for monitoring treatment and tumorprogression.

BACKGROUND

Retinoic acid induces the differentiation of many cell types, such asepithelial cells, mesenchyme cells, teratocarcinoma cells, leukaemiacells and immortalized cell lines such as embryonal carcinoma cells andneuroblastoma cells. RA is a morphogen which specifies axial patterningduring embryonic development and which affects neurogenesis, and hasbeen used as an effective therapeutic agent for the treatment of acutepromyelocytic leukaemia.

The exact mode of action of retinoic acid is currently unknown, althoughit is known to be mediated by the nuclear retinoic acid receptors (RARs)(Chambon, P., 1996, FASEB J., 10: 940-959), and it is hypothesised thatthe diverse effects of RA result from the differential regulation ofproteins such as transcription factors, enzymes and growth factorreceptors.

Cheung, W. M. W. et al. (1997, J. Neurochem., 68: 1882-1888) have usedRNA fingerprinting by arbitrarily primed PCR to identify a large numberof genes that are differentially regulated during RA-induced neuronaldifferentiation. The present inventors have succeeded in isolating,purifying and cloning a novel gene which is down-regulated duringRA-induced neuronal differentiation and whose resultant protein productpossesses morphogenic/mitogenic properties.

BRIEF SUMMARY

According to a first aspect of the present invention, there is providedan isolated nucleic acid of SEQ ID NO:1 encoding a retinoic acidregulated expression product having the amino sequence depicted in SEQID NO:2.

According to an additional aspect of the present invention, there isprovided an expression product of an isolated nucleic acid of SEQ IDNO:1. Suitably, the nucleic acid or the expression product thereof maybe used as a screening or supporting tool for the diagnosis ofHepatocellular carcinomas (HCC). The expression product may also beadapted for monitoring treatment or progression of Hepatocellularcarcinomas (HCC).

According to another aspect of the present invention, there is providedan antibody comprising the amino acid sequence depicted in SEQ ID NO:4that binds specifically to a retinoic acid regulated nuclear matrixprotein (RAMP) having an amino acid sequence depicted in SEQ ID NO:2.

The present invention is also directed to providing an antibodycomprising the amino acid sequence depicted in SEQ ID NO:5 that bindsspecifically to a retinoic acid regulated nuclear matrix protein (RAMP)having the amino acid sequence depicted in SEQ ID NO:2.

According to an additional aspect of the present invention, there isprovided a recombinant DNA construct comprising operatively linked insequence in the 5′ to 3′ direction: (i) a promoter region that directsthe transcription of a gene; (ii) a DNA coding sequence encoding an RNAsequence encoding an expression product having the sequence depicted inSEQ ID NO:2; and (iii) a 3′ non-translated region. In particular, theDNA coding sequence may comprise the sequence of SEQ ID NO:1. In otherembodiments of the present invention, there is provided a celltransformed or transfected with the recombinant DNA construct describedabove.

According to another aspect of the present invention, there is providedan isolated nucleic acid comprising a nucleotide sequence encoding theamino acid sequence depicted in SEQ ID NO:2.

In other embodiments of the present invention, there is provided amethod for screening and determining the prognosis of a patient havingHepatocellular cancer (HCC), the method comprising the steps of: (i)obtaining biological samples from the patient; (ii) isolating proteinsfrom the biological samples; (iii) contacting the proteins with aspecific antibody that binds specifically to a retinoic acid regulatednuclear matrix protein (RAMP) comprising the amino acid sequencedepicted in SEQ ID NO:2; and (iv) detecting the presence of anexpression product of SEQ ID NO:1 having the amino acid sequencedepicted in SEQ ID NO:2. The biological samples may comprise livertissues, while the antibody may be a polypeptide.

According to an additional aspect of the present invention, there isprovided a gene having the sequence depicted in SEQ ID NO:1. Alsoprovided is an expression product encoded by the gene of the presentinvention, and in particular an expression product of the gene havingthe sequence depicted in SEQ ID NO:2. The present invention also extendsto allelic mutants of the gene and gene expression product, and also tomodified forms of the nucleic acid sequence which encode the expressionproduct. For example, modifications may be made to the nucleic acidsequence such that it has a different sequence yet still codes for thesame amino acid sequence.

Experiments (described in more detail below) show that the expressionproduct is important in maintaining the stem cell identity of theprogenitor cells, as well as in the early differentiation of theprogenitor cells. It is also important in embryogenesis and also appearsto participate in the functioning of adult tissues, particularly brain,lung, liver and kidney. Expression of the gene product in lymphoidtissues shows a restrictive profile in the T-cell lineage of the immunesystem, particularly in the thymus and the bone marrow.

The gene of the present invention may also have applications in thetreatment of Ushers disease, particularly type II Ushers disease, andthus the present invention extends to the use of the gene and itsexpression product in the manufacture of medicaments for treating Ushersdisease, together with methods of treatment of Ushers disease.

Thus, the gene of the present invention is useful both in treating andpreventing diseases associated with its expression, with morphogeny andmitogeny, and with Ushers disease, particularly type II Ushers disease.

The expression product according to the present invention may be amitogen and/or a morphogen. Further, the expression product of thepresent invention may be usefully provided in the form of a recombinantconstruct, allowing its expression by chosen organisms under chosenconditions.

According to another aspect of the present invention, there is alsoprovided a DNA molecule, which may be in recombinant or isolated form,comprising a sequence encoding an expression product according to thepresent invention.

The coding sequence may be operatively linked to an expression controlsequence sufficient to drive expression. Recombinant DNA in accordancewith the invention may be in the form of a vector, for example aplasmid, cosmid or phage. A vector may include at least one selectablemarker to enable selection of cells transfected (or transformed) withthe vector. Such a marker or markers may enable selection of cellsharbouring vectors incorporating heterologous DNA. The vector maycontain appropriate start and stop signals. The vector may be anexpression vector having regulatory sequences to drive expression.Vectors not having regulatory sequences may be used as cloning vectors(as may expression vectors).

Cloning vectors can be introduced into suitable hosts (for example E.coli) which facilitate their manipulation. According to another aspectof the invention, there is therefore provided a host cell transfected ortransformed with DNA according to the present invention. Such host cellsmay be prokaryotic or eukaryotic. Expression hosts may be stablytransformed. Unstable and cell-free expression systems may of coursealso be used.

Expression hosts may contain other exogenous DNA to facilitate theexpression, assembly, secretion and other aspects of the biosynthesis ofmolecules of the invention.

The present invention may be used with synthetic DNA sequences, cDNAs,full genomic sequences and “minigenes”, i.e. partial genomic sequencescontaining some, but not all, of the introns present in the full-lengthgene.

DNA according to the present invention may be prepared by any convenientmethod involving coupling together successive nucleotides, and/orligating oligo- and/or polynucleotides, including in vitro processes, aswell as by the more usual recombinant DNA technology.

Also provided according to another aspect of the present invention is arecombinant DNA construct comprising operatively linked in sequence inthe 5′ to 3′ direction:

a) a promoter region that directs the transcription of a gene;

b) a DNA coding sequence encoding an RNA sequence encoding an expressionproduct of the present invention; and

c) a 3′ non-translated region.

The DNA coding sequence may have the sequence of SEQ ID NO:1.

Also provided is a cell transformed or transfected with a recombinantDNA construct of the present invention.

Also provided is a method of treating or preventing diseases associatedwith the expression of a gene of the present invention, comprisingadministering to a patient an expression product of the presentinvention.

As well as simply expressing the gene or administering the gene productin order to effect treatment of a patient, it may also be desirable toinhibit (i.e. antagonise) the gene product. This can be achieved in amultitude of ways, as will be readily apparent to one skilled in theart. In particular, U.S. Pat. No. 5,856,129 to Hillman, et al. and thereferences cited therein provide information regarding how to produceand identify antagonists, inhibitors and potentiators of gene products.U.S. Pat. No. 5,856,129 to Hillman, et al. is incorporated herein in itsentirety by reference thereto for all purposes. In particular, thefollowing additional teachings may be used: Harlow, E. and Lane, D.,“Using Antibodies: A Laboratory Manual”, Cold Spring Harbour LaboratoryPress, New York, 1998; Sambrook, J., Frisch, E. F., and Maniatis, T.,“Molecular Cloning. A Laboratory Manual”, Cold Spring HarbourLaboratory, Cold Spring Harbour Press, New York, 1989; Ausubel, F. M. etal., 1989, Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.; Gee, J. E. et al., 1994, In: Huber, B. E. and Can, B. I.Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.

The invention will be further apparent from the following descriptionwith reference to the figures, which shows by way of example only thecloning and study of the gene of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows coupled in vitro transcription and translation using rabbitreticulocyte extract, demonstrating that full length 8.31 cDNA encoded a˜80 kDa protein. Histidine (His)-tagged 8.31 protein was constructed bycloning 6 His to the C-terminus of 8.31. Coupled in vitro transcriptionand translation was performed in the absence of radioactive label. Thetranslated proteins were separated by SDS PAGE, transferred tonitrocellulose membrane and blotted with monoclonal antibody against the6×His tail;

FIG. 2 shows Northern blot analysis of 8.31 expression in RA-treated NT2cells. Total RNA (10n) prepared from NT2 cells treated with all-trans RAfor 0 to 28 days, separated by denaturing gel electrophoresis, andtransferred to nylon membrane. Hybridization was performed using thefull-length 8.31 cDNA as probe. Ribosomal RNA bands are as shown on theleft;

FIG. 3 shows the expression profile of 8.31 in human tissues. MultipleTissue Northern blots (Clontech) were hybridized using full-length 8.31cDNA as probe. Results of the hybridization using adult tissues (FIGS.3A and 3B) and fetal tissues (FIG. 3C) are shown. RNA size markers areindicated on the left;

FIG. 4 shows a dot blot analysis of 8.31 expression. Messenger RNA (2μg) was used in the dot blot to examine the expression of 8.31 invarious tissues of hematopoietic origin as well as fetal tissues.Results of the hybridization using full-length 8.31 cDNA as probe areshown. Adult cells (top and middle rows) are (left to right, top tobottom) small intestine, spleen, thymus, peripheral leukocyte, lymphnode, bone marrow, trachea and placenta. Fetal cells (bottom row) are(left to right) kidney, liver, spleen thymus and lung;

FIG. 5 shows RT-PCR analysis of the 8.31 expression in human cell lines.Total RNA (2 μg) obtained from neuronal precursor cell lines IMR32, andleukaemia cells was reverse transcribed and amplified by specificprimers for 8.31. KT4 represents treatment of KG1 cells with all-transRA for 4 days. Hybridization was performed to confirm the identity ofthe amplified products;

FIG. 6 shows expression of 8.31 in RA-treated HL-60 cells. Total RNA (15μg) from HL-60 cells treated with 0 to 6 days was used for Northern blotanalysis using full-length 8.31 cDNA probe. Ribosomal RNA bands areindicated on the left;

FIG. 7 shows chromosomal localization of the gene 8.31 by FISH. Gene8.31 was labeled and is shown marked “A”, and the specific marker forthe heterochromatin of chromosome I was labeled and so is shown marked“B”; and

FIG. 8 shows the expression of a gene product encoding for RAMP in thetumor of a HCC patient. The gene products encoding for RAMP from proteinextracts of a HCC patient is shown as “N” for normal tissues adjacent tothe tumor (FIG. 8B); “T” for tumor samples (FIG. 8B); “U” forundifferentiated NT2 cells used to normalize the expression of RAMPbetween blots (FIG. 8A) and “D” for differentiated NT2 cells by retinoicacid (FIG. 8A).

EXAMPLES Example I

Hepatocellular carcinoma (HCC) is the most common primary cancer of theliver. (Parkin D M, Whelan S L, Ferlay J, et al., eds.: Cancer Incidencein Five Continents: Volume VII. Lyon, France: IARC ScientificPublications, pp. 1072-1074, 1997). In the United States, it isestimated that there will be 17,300 new cases in liver and intra hepaticbile duct cancer and 14,400 deaths in 2003 (American Cancer Society:Cancer Facts and FIGS. 2003. Atlanta, Ga.: American Cancer Society, p.4, 2003). Over the past two decades, the number of cases of HCC in theUnited States increased substantially and the age-specific incidence ofthis cancer has progressively shifted towards younger people with agebetween 40 to 60 years old (EL-Serag H B, Mason A C. Rising Incidence ofHepatocellular Carcinoma in the United States. The New England Journalof Medicine 340 (10): 745-750, 1999).

Alterations in nuclear morphology are hallmarks of cancer and arebelieved to be associated with changes in nuclear matrix composition.Nuclear matrix provides structural support for the nucleus and plays adynamic role in the spatial organization of the genome and in thecontrol of DNA replication and transcription. The recovery of increasedamount of specific nuclear matrix proteins (NMP) in several differentcancers has led to the further study of some of these proteins as a newclass of tumor markers (Keesee S K, Briggman J V, Thill G. Wu Y J:Utilization of Nuclear Matrix Proteins for Cancer Diagnosis. CriticalReviews in Eukaryotic Gene Expression 6 (2&3): 189-214, 1996). Recently,specific nuclear matrix proteins have been isolated and weredemonstrated to have prognostic value: NMP22 as a marker fortransitional cell carcinoma of urinary bladder (Chahal R. Darshane A,Browning A J, Sundaram S K: Evaluation of the clinical value of urinaryNMP22 as a marker in the screening and surveillance of transitional cellcarcinoma of the urinary bladder. Eur Urol. 40: 415-20, 2001); NMP66 asa marker for breast cancer (Luftner D, Possinger K: Nuclear matrixproteins as biomarkers for breast cancer. Expert Rev Mol Diagn 2(1):23-31, 2002); NMP survivin expression in oesophageal squamous cellcarcinoma (Grabowski P, Kuhnel T, Muhr-Wilkenshoff F, Heine B, Stein H,Hopfner M, Germer C T, Scherubl H: Prognostic value of nuclear survivinexpression in oesophageal squamous cell carcinoma. Br J Cancer 88:115-9, 2003); and c-myc as a marker for melanoma (Chana J S, Grover R,Tulley P, Lohrer H, Sanders R, Grobbelaar A O, Wilson G D: The c-myconcogene: use of a biological prognostic marker as a potential targetfor gene therapy in melanoma. Br J Plast Surg 55: 623-7, 2002).

The present invention identified a novel gene comprising an isolatednucleic acid sequence according to SEQ ID NO:1, the expression productof which, a novel retinoic acid regulated nuclear matrix protein (RAMP),is detected in over 70% of patient samples at the early stages of HCCwhile the normal liver tissue adjacent to the tumor tissue has showed avery low or undetectable expression of RAMP. Immunoreactive bandsdetected by the antibody recognizing RAMP were at about 80 kDa in tumorsamples (FIG. 8). Intensity of the RAMP immunoreactive protein bands wasquantitated by densitometry. In tumors, an overexpression of RAMP wasdetermined when there was an at least twofold increase in theintensities of the immunobands compared to adjacent normal tissues.Specifically, 22 out of 28 carcinomas revealed an overexpression of theRAMP. Of these RAMP overexpressing tumors, 16 samples exhibited anadditional lower molecular weight RAMP band, migrating to about 60 kDa(FIG. 8). In some of the tumors the lower molecular weight isoforms wereeven more abundant than the higher molecular weight protein of RAMP(FIG. 8).

FIG. 8 is concerned with the expression of a gene product encoding forRAMP in the tumor of a HCC patient. Examples of a representative Westernblot analysis was used to show the expression of gene products encodingfor RAMP from protein extracts of a HCC patient (N, normal adjacenttissues; T, tumor samples). Protein (20 μg) was loaded to each lane. Theexpression of gene product for RAMP in undifferentiated NT2 cells (U)was used to normalize the expression of RAMP between blots.

Materials and Methods:

Hepatocellular carcinomas (n=28) and matched adjacent normal livertissues (n=28) were obtained. The tissues were stored at −80° C. beforeprocessing. The proteins were extracted from tumor and adjacent normaltissues of different individuals. Protein concentration of the lysateswas determined using Bio-Rad Protein Assay kit (Bio-Rad). The proteinwas then resuspended in sample buffer. The composition of the samplebuffer was 0.125 M Tris-HCl buffer (pH 6.8), 4% SDS, 10%β-mercaptoethanol, 20% glycerol and 0.002% bromophenol blue. The mixturewas boiled for 3 min and then the supernatant was loaded in theSDS-polyacrylamide gel.

Equal amount of protein was separated on SDS-PAGE gels. SDS-PAGE wascarried out using a 7.5% acrylamide resolver gel and 4% stacker gelaccording to Laemmli et al. (1970). The stacker and resolver gels wereprepared according to the protocol supplied with the electrophoresisapparatus, Mini-Protein II (Hoefer, Amersham Biosciences). The sampleswere loaded into the wells of a 1.0 mm thick gel and electrophoresedwith 20 mA through the stacker gel and 30 mA after entering the resolvergel for 3 hr in 25 mM Tris-HCl and 192 mM glycine (pH 8.3), containing0.1% SDS. Prestained molecular weight markers and protein sample ofundifferentiated NT2 cells were run alongside the samples.

The proteins on the polyacrylamide gel were transferred onto anitrocellulose membrane in 1× transfer buffer using a Trans-Blotelectrophoretic transfer cell (Bio-Rad. CA, USA) at 100 V for 1 hr at 4°C. The membrane was washed with Tris-buffered saline with 0.1% Tween-20(TBS-T). The membrane was blocked with 5% non-fat dry milk in TBS-T for1 hr at room temperature. The membrane was then incubated with RAMPantibody (1:500) in 1×TBS-T with 5% BSA at 4° C. overnight, followedwith horseradish peroxidase conjugated secondary antibodies in TBS-Tweenwith 5% nonfat milk at room temperature for 1 hr. The immunoreactiveproteins were detected using Pico detection system (Pierce, Rockford,USA) according to the supplier's instruction.

Example II

The gene of the present invention (also referred to as clone 8.31) wascloned and expressed, its in vitro transcription and translation assayedand its chromosomal location determined. The expression profile of 8.31in a range of cell types and under a range of conditions has allowed arole for it to be determined.

Materials and Methods:

Experimental methods referred to and used are standard laboratorytechniques. Where specific methods are not described or referenced, fulldescriptions and protocols are well known in the art and available inlaboratory manuals such as Harlow, E. and Lane, D., “Using Antibodies: ALaboratory Manual”, Cold Spring Harbor Laboratory Press, New York, 1998;Sambrook, J., Frisch, E. F., and Maniatis, T., “Molecular Cloning. ALaboratory Manual”, Cold Spring Harbor Laboratory, Cold Spring HarborPress, New York, 1989; PCR (Volume 1): A practical approach. Eds. M. J.McPherson, P. Quirke and G. R. Taylor. Oxford University Press, 1991;and Torres, R. M. and Kuhn, R., “Laboratory Protocols for ConditionalGene Therapy”, Oxford University Press, 1997, ISBN 019963677-X.

Cloning of Full Length cDNA of 8.31:

Full length cDNA of 8.31 was obtained by screening an expression cDNAlibrary prepared from undifferentiated NT2 cells (STRATAGENE) using thepartial 8.31 cDNA fragment (SEQ ID NO:3) as probe. Radioactive cDNAprobes were prepared using the Megaprime DNA labelling system(AMERSHAM). Single phages were obtained and transformed into XLOLRbacterial cells (STRATAGENE) and the cDNA fragment cloned into pBK-CMVmammalian expression vector by in vivo excision.

Cell Culture:

NT2 cells were cultured as previously described (Cheung et al., 1996,NeuroReport, 6: 1204-1208). Cells were maintained in Opti-MEM Ireduced-serum medium (GIBCO) supplemented with 5% fetal bovine serum(FBS, GIBCO). NT2 cells were differentiated with 5 μM all-trans RA(SIGMA) in Dulbecco's modified Eagle's medium (DMEM; high glucoseformulation) supplemented with 10% FBS. Leukaemia cell lines werecultured as previously described (Xie et al., 1997, NeuroReport, 8:1067-1070).

8.31 cDNA Probe:

The partial cDNA sequence of 8.31 was obtained using RNA fingerprintingby arbitrarily primed PCR (RAP-PCR, Welsh, J. et al., 1992, NucleicAcids Res., 20: 4965-4970). Total RNA was obtained from NT2 cellstreated for various durations with all-trans RA (10 M). Thedifferentially-regulated cDNA fragments were cloned into pCRscript SK+for DNA sequencing. The cDNA probe (SEQ ID NO:3) was then used to screenan undifferentiated human NT2 cell cDNA library for the full length 8.31cDNA.

RNA Preparation, RT-PCR and Northern Blot Analysis:

Total RNA was prepared using Trizol reagent (GIBCO) or as previouslydescribed (Xie et al., 1997, NeuroReport, 8: 1067-1070). Equal amountsof total RNA from different cell lines were used for Northern blotanalysis, while 2 μg total RNA was used for reverse transcription usingSuperscript II reverse transcritptase (GIBCO). One tenth of the reactionwas amplified using Taq DNA polymerase (GIBCO). Gene expression wasconfirmed by using different numbers of PCR cycles and hybridizationusing 8.31 specific cDNA probes.

Coupled In Vitro-Transcription and Translation:

Two micrograms of plasmids were used for each coupled in vitrotranscription/translation reaction using the TNT coupled reticulocytelysate system (PROMEGA).

Chromosomal Localization of 8.31 by FISH (Fluorescent In SituHybridisation):

Genomic DNA encoding 8.31 was labeled with digoxigenin (DIG) dUTP bynick translation and was hybridized to normal metaphase chromosomesderived from PHA phytohemaglutinin stimulated peripheral bloodlymphocytes. After incubation with fluorescein-conjugated anti-DIGantibodies, the cells were counterstained with DAPI(4,6-diamidino-2-phenylindole), a fluorescent DNA groove-binding probe.

Results: Cloning of Full Length Coding Sequence of 8.31:

The cDNA encoding the full length 8.31 was obtained from a cDNA libraryprepared from undifferentiated NT2 cells using hybridization screening.Double stranded sequencing by T7 DNA polymerase revealed that the cDNA(˜2831 bp) is novel in its gene identity (FIG. 1). The coding sequencecan be translated into a protein of 730 amino acid residues. Coupled invitro transcription and translation was performed to demonstrate thatthe cloned cDNA can be translated into a protein with molecular weightof ˜80 kDa (FIG. 1).

Transcript Expression of 8.31:

The full length 8.31 was then used as a probe to examine its expressionwhen the NT2 cells were treated with RA for 0 to 28 days (FIG. 2). Twotranscripts were obtained (−4.5 kb and ˜3.5 kb). The expression of 8.31was slightly induced after 1 day of RA treatment. At day 2, theexpression decreased to its basal level and then continue to decreasealong the course of RA treatment. Its expression was almost halted atday 28.

To obtain clues on the potential functions of 8.31, we examined theexpression profile of 8.31 in both adult and fetal human tissues. Amongthe adult tissues examined, which include heart, brain, placenta, lung,liver, skeletal muscle, kidney, pancreas, stomach, and testis, prominentexpression of 8.31 was observes in placenta and testis. Skeletal musclesexpressed low levels of 8.31 (FIG. 3, panels A and B).

The expression of 8.31 was observed in all the human fetal tissuesexamined, which included brain, lung, liver and kidney. An extratranscript (˜5.5 kb) was observed in all fetal tissues and a smalltranscript (˜2.4 kb) was observed only in the messenger RNA preparedfrom the fetal lung (FIG. 3, panel C). The high expression of 8.31 inthe fetal tissues examined was not observed in the corresponding adulttissues.

Dot blot analysis was performed to examine the expression of 8.31 inhematopoietic tissues. Expression of 8.31 was detected in allhematopoietic tissues examined; however, 8.31 was predominantlyexpressed in the thymus and the bone marrow. Lower transcript expressionof 8.31 was detected in the spleen and lymph node. Only a barelydetectable level of its expression was observed in the peripheralleukocytes (FIG. 4).

Expression of 8.31 in Different Human Cell Lines:

Owing to the high expression of 8.31 detected in the hematopoietictissues, we have examined its expression in several leukaemia cell linesto obtain clues on its roles in hematopoietic systems. RT PCR analysiswas performed using total RNA prepared from K562, KG1, HL-60, HL-60S4,and CEM, cell lines each corresponding to a different type of leukaemia(FIG. 5). Transcript expression of 8.31 was observed in allhematopoietic cell lines tested. Its expression was also observed in ahuman neuroblastoma cell line, IMR32 cells.

Expression of 8.31 was Down-Regulated in RA-Treated HL-60 and KG1 Cells:

HL-60 cells were differentiated with 1 μM all-trans RA and theexpression of 8.31 was examined by Northern blot analysis. Twotranscripts (˜5.5 kb and ˜3.5 kb) were detected in undifferentiatedHL-60 cells (FIG. 6). When HL-60 cells were treated with RA for 3 days,its expression was significantly down-regulated (FIG. 6). At day 6 of RAtreatment, the expression of 8.31 was diminished.

The expression of 8.31 was also down-regulated when KG1 cells weretreated with 1 μM all-trans RA, as demonstrated by the RT-PCR analysis(FIG. 5).

Chromosomal Localization of Clone 8.31 by Fluorescence In SituHybridization:

DNA from a genomic clone of 8.31 was labeled with digoxigenin dUTP bynick translation. Labeled probe was combined with sheared human DNA andhybridized to normal metaphase chromosomes derived from PHA stimulatedperipheral blood lymphocytes. The initial experiment resulted inspecific labelling of the long arm of a group A chromosome which wasbelieved to be chromosome 1 on the basis of size, morphology, andbanding pattern. A second experiment was conducted in which abiotin-labelled probe specific for the heterochromactic region ofchromosome 1 was co-hybridized with the genomic clone of 8.31. Thisexperiment resulted in a specific labeling of the heterochromatin in red(marked “B” in FIG. 7) and the long arm in green (marked “A” in FIG. 7)of the chromosome 1. Measurements of 10 specifically labeled chromosomes1 demonstrated that the genomic clone of 8.31 is located at a positionwhich is 62% of the distance from the heterochromatic-euchromaticboundary to the telomere of chromosome arm 1q, an area which correspondsto band 1q32.1-32.2 (FIG. 7). A total of 80 metaphase cells wereanalyzed with 76 exhibiting specific labelling.

Type II Ushers syndrome (classical retinitis pigmentosa combined withcongenital pedial deafness, and normal vestibular function) has beenmapped to the chromosomal region containing the gene of the presentinvention (Kimberling et al., 1990, Genomics, 2: 245-249); Lewis et al.,1990, Genomics, 2: 250-256) and it appears that the gene of the presentinvention, together with its expression product, may be useful in thetreatment of Ushers syndrome. For example, the lack of functionresulting from mutations in the diseased gene may be complemented by thegene and/or expression products of the present invention.

Functional Roles of 8.31:

The expression profile observed for 8.31 suggests a potential role intissues of hematopoietic origin. Recently, placental blood has been usedas a rich source of hematopoietic stem cells for transplantation. Takentogether with the high expression of 8.31 in the testis and theundifferentiated NT2 cells, the expression of 8.31 in placenta revealeda strong association of the gene to the identity of the stem cells.Hence it appears that the gene product of 8.31 is important inmaintaining the stem cell identity of the progenitor cells, as well asin the early differentiation of the progenitor cells.

The expression of 8.31 is also strongly associated with the earlyembryonic development. This is exemplified by the high expression of8.31 in fetal tissues such as brain, lung, liver and kidney, but not insame adult tissues. Together with its restrictive expression pattern inthe adult tissues, it appears that the gene product of 8.31 is not onlyimportant in the embryogenesis, but is also participates in thefunctioning of these adult tissues. Different 8.31 isoforms exist, theexpression of which can be regulated during the development (FIG. 3).

The predominant expression of 8.31 in the thymus and the bone marrow,but low expression in other lymphoid tissues revealed its restrictivefunctions in the T-cell lineage of the immune system.

Involvement of 8.31 in the Differentiation of Cancer Cells:

Northern blot analysis demonstrated the down-regulation of 8.31expression with the treatment of all-trans RA. HL-60 is an acutepromyelocytic leukemia cell line. The growth rate was sharply decreasedby treatment with RA. It appears that the expression of 8.31 is stronglyassociated with the differentiation of other cancer cell lines,including the embryonal carcinoma cells and the neuroblastoma cells.Hence 8.31 may serve as a diagnostic marker for different cancer types.

8.31 as a Candidate Gene for Genetic Diseases:

The gene encoding 8.31 was localized to the chromosome 1q32.1 32.2Chromosome 1q 32 locus has been mapped to several genetic diseasesincluding the complement system malfunctioning, as well as the Usherdisease, which is related to hearing. Moreover the Alzheimer's diseaseis also mapped to the region 1q32, although the exact position remainsto be elucidated.

Unless stated otherwise, all procedures were performed using standardprotocols and following manufacturer's instructions where applicable.Standard protocols for various techniques including PCR, molecularcloning, manipulation and sequencing, the manufacture of antibodies,epitope mapping and mimotope design, cell culturing and phage display,are described in texts such as McPherson, M. J. et al. (1991, PCR: Apractical approach, Oxford University Press, Oxford), Sambrook, J. etal. (1989, Molecular cloning: a laboratory manual, Cold Spring HarbourLaboratory, New York). Huynh and Davies (1985, “DNA Cloning Vol 1-APractical Approach”, IRL Press, Oxford, Ed. D. M. Glover), Sanger, F. etal. (1977, PNAS USA 74(12): 5463-5467), Harlow, E. and Lane, D. (“UsingAntibodies: A Laboratory Manual”, Cold Spring Harbour Laboratory Press,New York, 1998), Jung, G. and Beck-Sickinger, A. G. (1992, Angew. Chem.Int. Ed. Eng., 31: 367-486), Harris, M. A. and Rae, I. F. (“GeneralTechniques of Cell Culture”, 1997, Cambridge University Press, ISBN 0521573645), “Phage Display of Peptides and Proteins: A Laboratory Manual”(Eds. Kay, B. K., Winter, J. and McCafferty, J., Academic Press Inc.,1996, ISBN 0-12-402380-0). Reagents and equipment useful in, amongstothers, the methods detailed herein are available from the likes ofAmersham, Boehringer Mannheim, Clontech, Genosys, Millipore, Novagen,Perkin Elmer, Pharmacia, Promega, Qiagen, Sigma and Stratagene.

What is claimed is:
 1. An isolated antibody to SEQ ID NO:2.
 2. A methodof inhibiting activity of an expression product having SEQ ID NO:2, themethod comprising providing an antibody according to claim 1, andcontacting the expression product with said antibody.